ctDNA Status Research Articles

Phase II/III study of circulating tumor DNA as a predictive biomarker in adjuvant chemotherapy in patients with stage II colon cancer: NRG-GI005 (COBRA).

TPS3625 Background: Detection of circulating tumor DNA (ctDNA) shed into the bloodstream represents a highly specific and sensitive approach for identifying microscopic or residual tumor cells after surgical resection. For patients (pts) with colon cancer (CC), the detection of ctDNA is associated with persistent disease after resection and outperforms traditional clinical and pathological features in prognosticating risk for recurrence. However, for pts with stage II CC, there are currently no validated biomarkers predicting benefit in identifying pts whose residual disease cancer be cleared by adjuvant chemotherapy. We hypothesize that for pts whose stage II colon cancer has been resected and who have no traditional high-risk features, a positive ctDNA status may identify those who will benefit from adjuvant chemotherapy. Methods: In this prospective phase II/III clinical trial, pts (N = 1,408) with resected stage II CC without traditional high-risk features and whom the evaluating oncologist deems suitable for active surveillance (i.e., not needing adjuvant chemotherapy) will be randomized 1:1 into 2 arms: standard-of-care/observation (Arm A), or prospective testing for ctDNA (Arm B). Postoperative blood will be analyzed for ctDNA with the Guardant Reveal assay, covering CC-relevant mutations and CC-specific methylation profiling. Pts in Arm B with ctDNA detected will be treated with 6 months of adjuvant (FOLFOX) chemotherapy. For all pts in Arm A, ctDNA status will be analyzed retrospectively at the time of endpoint analysis. The primary endpoints are clearance of ctDNA with adjuvant chemotherapy (phase II) and recurrence-free survival (RFS) for “ctDNA-detected” pts treated with or without adjuvant chemotherapy (phase III). Secondary endpoints will include time-to-event outcomes (OS, RFS, TTR) by ctDNA marker status and treatment, prevalence of detectable ctDNA in stage II CC, and rates of compliance with assigned intervention. Archived normal and matched tumor and blood samples will be collected for exploratory correlative research. Enrollment continues across North America to the 1408-patient phase III endpoint. NCT#: 04068103. Support: U10CA180868, -180822; UG1CA189867; GuardantHealth. Clinical trial information: NCT04068103 .

Circulating tumor DNA (ctDNA) status and clinical outcomes in patients (pts) with previously untreated diffuse large B-cell lymphoma (DLBCL) in the POLARIX study.

7523 Background: the POLARIX study, pts with previously untreated DLBCL were randomized to Pola-R-CHP or R-CHOP (NCT03274492; Tilly et al. 2022). We previously validated the prognostic value of ctDNA at baseline and after one cycle of therapy in POLARIX (Herrera et al. 2022); here, we evaluate the relationship between ctDNA clearance and PFS and OS. Methods: Pts were included if baseline and longitudinal ctDNA results were available. Plasma ctDNA was measured at baseline, Cycle (C) 5 Day (D) 1, and end of treatment (EOT), with the AVENIO NHL CAPP-Seq assay (Stokowski et al. 2022). ctDNA clearance was determined as previously described (Herrera et al. 2022). PFS and OS according to ctDNA status were reported as landmarked hazard ratios (HR) and 3-yr rates. HRs were adjusted for IPI score (>2), region, bulky disease (>7.5cm), age (>60 years), and cell of origin. Results: At baseline, 654 pts had ctDNA results; 494 (76%) and 519 (79%) pts were evaluable at C5D1 and EOT, respectively. Undetectable ctDNA (ctDNA−) was achieved by 57% (152/265) of Pola-R-CHP-treated pts and 59% (135/229) of R-CHOP-treated pts at C5D1 (p=0.79), and by 66% (172/262) of Pola-R-CHP-treated pts and 67% (172/257) of R-CHOP-treated pts at EOT (p=0.83). Achieving ctDNA− was prognostic for PFS and OS in each treatment arm at C5D1 and EOT (Table). Pts in the Pola-R-CHP arm who had complete response (CR) with PET-CT and ctDNA− at EOT had superior PFS and OS compared with pts with CR and detectable ctDNA (ctDNA+) at EOT (PFS HR 0.30, 95% confidence interval [CI]: 0.14–0.66; OS HR 0.20, 95% CI: 0.07–0.60). This was not observed with R-CHOP (PFS HR 0.74, 95% CI: 0.34–1.63; OS HR 1.11, 95% CI: 0.30–4.16). Among pts with CR and ctDNA− at EOT, pts treated with Pola-R-CHP had superior PFS vs R-CHOP (HR 0.41, 95% CI: 0.21–0.82); this was not observed with OS (HR 0.41, 95% CI: 0.14–1.18). There was no statistically significant difference in PFS between treatment arms in pts with CR and ctDNA+ at EOT (PFS HR 1.20, 95% CI: 0.49–2.83). Conclusions: Achieving ctDNA− at C5D1 and EOT was prognostic for longer survival. Although no difference was observed in the number of pts with ctDNA− at EOT between arms, pts achieving CR and ctDNA− at EOT had superior PFS in the Pola-R-CHP vs the R-CHOP arm, suggesting deeper molecular responses in pts treated with Pola-R-CHP beyond the detection sensitivity of the assay used in this analysis. Clinical trial information: NCT03274492 . [Table: see text]

Circulating tumor DNA dynamics as an early predictor of recurrence in patients with radically resected colorectal cancer: Updated results from GALAXY study in the CIRCULATE-Japan.

3521 Background: Postoperative circulating tumor DNA (ctDNA)-based molecular residual disease (MRD) is reported to be associated with a high risk of recurrence. Here, we present an updated analysis and the lead time interval (LTI) of ctDNA positivity to radiographic recurrence in patients (pts) with radically resected colorectal cancer (CRC), stage II-IV in the observational GALAXY study. Methods: Serial ctDNA was analyzed using personalized tumor-informed assay (Signatera bespoke multiplex-PCR NGS assay) at 1 (4-week MRD time point), 3, 6, 9, 12, 18, and 24 months after surgery until recurrence, and CT scans were conducted every 6 months. The primary endpoint was disease-free survival (DFS), defined as the time between the date of surgery and date of diagnosis with relapse or death due to any cause. The LTI was defined from the first ctDNA positive date at post-surgery to radiographic recurrence. Results: Among 3,615 CRC pts who were enrolled between May 2020 and April 2022 in GALAXY study, 2,083 pts that met the inclusion criteria were analyzed in this report. The median follow-up period was 16.3 months. Of 2,083 pts analyzed, 286 (14%) were ctDNA-positive at 4-weeks MRD time point and 1,797 (86%) were ctDNA-negative. Pts with ctDNA-positivity at 4-weeks MRD timepoint demonstrated an inferior DFS and were 12 times more likely to recur, compared to ctDNA-negative pts (HR:12, 95CI: 9.1-15%; p < 0.0001). We further combined ctDNA status with BRAF V600E status at 4-week MRD time point and observed that pts with ctDNA-positivity and BRAF V600E mutation showed significantly shorter DFS compared to ctDNA-positive pts with BRAF wild-type (p < 0.001). Whereas, in pts with ctDNA-negativity no significant difference in DFS was observed (p = 0.306). On performing ctDNA dynamics analysis between 4-weeks to 12 weeks, compared to pts who remained ctDNA-negative (N = 1529), a significantly shorter DFS was observed for pts who converted from ctDNA-negative to positive (N = 112, HR: 14.5, 95%CI: 8.8-23.8%, p < 0.0001) or who remained positive (N = 124, HR: 25.4, 95%CI: 18.3-35.3; p < 0.0001). Radiographic recurrence was observed in 186 pts (46%). Of these, 121 pts (65%) had CT imaging performed every 6 months, as per the protocol. The median LTI was 142 days (IQR, 43-189 days). Conclusions: Our study builds on the existing evidence from the recently published, prospective GALAXY study. ctDNA status at MRD time point post-surgery is prognostic of patient outcomes and is the most significant risk factor regardless of BRAF V600E status. ctDNA positivity predicted radiologic recurrence several months ahead of clinical recurrence. Pts with positive postoperative ctDNA should be examined carefully due to a high risk of recurrence. ctDNA-guided adjuvant strategy will further be established by ongoing randomized VEGA and ALTAIR studies in the CIRCULATE-Japan. Clinical trial information: UMIN000039205 .

Accuracy of genome-wide mutational analysis for tumor-informed ctDNA-guided MRD monitoring in bladder cancer.

4589 Background: Neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) is the standard management of localized muscle-invasive bladder cancer (MIBC). However, about 45% of patients develop metastases within 2 years after RC. Biomarkers for treatment efficacy evaluation and early detection of minimal residual disease (MRD) are needed for earlier treatment initiation and monitoring of treatment response. Tumor-informed detection of mutations in cell-free DNA (cfDNA) has shown promising results to monitor MRD. However, the low tumor fraction after surgery and limited input material obtained from a typical plasma sample limits the probability of detecting low metastatic burden scenarios. Here we implemented and applied a whole-genome sequencing (WGS) approach to monitor circulating tumor DNA (ctDNA) for sensitive ctDNA detection. Methods: 110 MIBC patients undergoing NAC and RC were enrolled. cfDNA was extracted from ~1mL plasma (n=978) and procured from longitudinal plasma sampling during NAC and pre-RC and post-RC. WGS of tumor/germline pairs (30x/20x) and plasma cfDNA (>20x) was performed, facilitating detection of genome-wide genomic alterations and quantification of ctDNA using the C2inform method. Results: For each patient we developed a tumor-informed WGS model by integrating genome-wide mutation and copy number variation data coupled with advanced signal processing and AI-based error suppression. Patient-specific somatic variant patterns were used for detection and measuring ctDNA levels in low-input blood samples by WGS. Post-RC ctDNA analysis identified patients with recurrence with 78% sensitivity and 95% specificity and with a median lead time over radiographic imaging of 118 days (full follow-up included). When restricting follow-up time to 12 months following the latest ctDNA test, a sensitivity of 86% and specificity of 94% was achieved. ctDNA status was associated with recurrence-free (p<0.0001) and overall survival (p<0.0001). Furthermore, ctDNA clearance during NAC was also associated with recurrence-free survival (p=0.0051). The APOBEC associated signatures SBS2 and SBS13 were identified as the primary contributors to the mutational landscape of the primary tumors and TP53, KMT2D, PIK3CA, RB1 and KDM6A were the main affected driver genes. Half of the tumors displayed whole genome doubling. Moreover, our analysis of plasma samples after treatment showed the presence of chemotherapy-induced mutational signatures not present in the primary tumor, along with increases in copy numbers on chromosomes 19q and 20, and a focal amplification of the FGFR3 gene on chromosome 4. Conclusions: Our results highlight the clinical potential of personalized genome-wide mutation integration as an ultra-sensitive, non-invasive method for MRD detection and treatment response monitoring.

Longitudinal circulating tumor DNA monitoring in patients with esophageal squamous cell carcinoma.

3041 Background: The clinical utility of longitudinal monitoring with circulating tumor DNA (ctDNA) as a prognostic and a predictive biomarker was recently reported in patients with resectable colorectal cancer (Kotani D, et al. Nature Medicine 2023). Here we report a prospective, observational study of longitudinal ctDNA monitoring in patients with esophageal squamous cell carcinoma (ESCC) receiving neoadjuvant chemotherapy (NAC) followed by surgery. Methods: Patients with ESCC planned for NAC followed by curative surgery, were prospectively enrolled in this study. As per the protocol, plasma samples were collected at 7 timepoints: pre-NAC, post-NAC, and 4,12, 24, 36, and 48-weeks after surgery. CT scans were conducted every 3 months. Longitudinal ctDNA testing was performed using a personalized, tumor-informed ctDNA assay (Signatera, bespoke mPCR, NGS assay). In this initial report, ctDNA results at 3 time points (pre-NAC, post-NAC, and 4 weeks post-surgery) were analyzed and their association with clinical outcomes and tumor regression grade (TRG) scores were assessed. Results: So far, a total of 83 blood samples from 28 patients with ESCC were collected serially at pre-NAC (N=28), post-NAC (N=27), and post-surgical (N=28) time points. Pre-NAC ctDNA was detectable in 96.4% patients (27/28), and detection rates decreased at the post-NAC (37%; 10/27) and post-surgical (11%; 3/28) timepoints. ctDNA-positivity post-NAC and post-surgery was associated with a significantly shorter recurrence free survival (RFS) with HR of 7.0 (95% CI: 1.4-35.0; p = 0.02) and HR of 14 (95% Cl 3.0-62; p <0.001), respectively. Notably, patients who achieved ctDNA clearance with NAC had favorable RFS as compared to the patients who did not (HR=0.15, 95% CI: 0.03 – 0.75; p = 0.02). Post-NAC ctDNA was also associated with tumor regression grade (TRG) score. None of the patients with TRG3 score (N=3) were ctDNA positive, whereas 7.7% (1/13) patients in the TRG2 category and 81.8% (9/11) in the TRG1 category were ctDNA-positive. Conclusions: Our study demonstrates a strong correlation between ctDNA status and clinical outcomes in ESCC patients. Assessment of ctDNA may be useful monitoring in patients with ESCC post NAC/surgery and may be used in the future to direct post-surgical management in this patient population. Clinical trial information: UMIN000042003 .

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: While nearly all younger patients (pts) with Hodgkin's lymphoma (HL) are cured with conventional chemotherapy, outcomes are significantly worse in those over age 60. Intriguingly, among older HL pts, those with EBV+ disease have even poorer outcomes (Keegan et al., JCO 2005), yet the biological basis for this phenomenon is not understood. The paucity of malignant cells in HL tumors has hampered genomic studies. Circulating tumor DNA (ctDNA) profiling can overcome these limitations, allowing noninvasive genotyping and risk stratification in HL (Spina et al., Blood 2018; Alig et al., ASH 2022). Here, we use a fully non-invasive approach for genomic profiling of elderly HL pts, focusing on the impact of ctDNA and EBV status on clinical outcomes. Methods: We profiled 57 pts (64%) from NCT02414568, a prospective, multicenter phase II LYSA-PVAB study of newly diagnosed classical HL >60 years (Ghesquières et al., ASH2019). All pts received 6 cycles of PVAB (prednisone, vinblastine, doxorubicin, and bendamustine). Plasma cfDNA was genotyped using Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq); MRD was monitored using Phased variant Enrichment and Detection Sequencing (PhasED-Seq). Pts were classified into high and low pretreatment ctDNA levels using a threshold of 2.5 log hGE/mL (Alig et al., ASH2022). We defined EBV status either by LMP1 staining of FFPE tumor tissue or by VirCAPP-Seq targeting 180 viral species, where a predefined threshold of 32 genomes/ml was previously validated as 94% accurate (Garofalo et al., ASH 2022). Results: Pretreatment ctDNA was detected in 94% of pts with a median concentration of 2.2% (MAF), which was significantly correlated with disease burden measured by TMTV (p = 0.034). Pts with high pre-treatment ctDNA levels (39%) had significantly worse PFS (HR = 2.3, p = 0.039). EBV+ pts also had inferior PFS (HR = 2.2, p = 0.04) and OS (HR = 3.1, p = 0.028) (Figures A-B). In multivariate analysis, both indices remained independently associated with worse outcomes (HR = 2.5, p = 0.01 for pre-treatment ctDNA and HR = 2.7, p = 0.01 for EBV status). Interestingly, pre-treatment ctDNA levels did not differ between EBV+ and EBV- tumors (p = 0.92), while specific genetic aberrations were significantly associated with respective EBV status (Figure C). Among evaluable pts for MRD monitoring, detection of ctDNA after four cycles of chemotherapy was highly associated with risk of death (Figure D). The research was funded by: NIH/NCI R01 R01CA257655 Keywords: Diagnostic and Prognostic Biomarkers, Genomics, Epigenomics, and Other -Omics, Hodgkin lymphoma Conflicts of interests pertinent to the abstract. C. Rossi Consultant or advisory role: Genmab, Janssen, Incyte Educational grants: Kyte, Abbvie S. K. Alig Consultant or advisory role: Takeda Pharmaceuticals D. M. Kurtz Consultant or advisory role: Adaptive Biotechnologies, Roche, Foresight O. Casasnovas Consultant or advisory role: Roche, Takeda Educational grants: Roche, Takeda, BMS, Janssen A. A. Alizadeh Consultant or advisory role: Adaptive Biotechnologies, Foresight Diagnostics, Roche, BMS, Gilead, Genentech, Karyopharm

Dynamic ctDNA to inform the precise management of resected NSCLC: LUNGCA-2 study.

8528 Background: Circulating tumor DNA (ctDNA) has been reported to be valuable for minimal residual disease (MRD) detection, but there is a lack of studies comprehensively assessing whether dynamic ctDNA monitoring can inform whole-course precise postoperative cancer management. In the present study (LUNGCA-2), we analyzed data obtained from longitudinal ctDNA monitoring of stage I-III NSCLC patients in the LUNGCA cohort, and sought to assess the use of dynamic ctDNA monitoring for precise whole-course postoperative management of NSCLC patients. Methods: All eligible NSCLC patients underwent curative-intent surgery and subsequently received adjuvant therapies when indicated by clinical practice guidelines for NSCLC. The clinical follow-up was scheduled to be every 3-6 months mainly involving CT and/or MRI. Fresh frozen or paraffin-embedded tissues of primary tumors were collected intraoperatively for baseline somatic mutation profiling. Blood samples were collected before surgery, at 3 days and 1 month after surgery, and then every 3-6 months at clinical visits up to 3 years postoperatively. 233 patients with a total of 2123 plasma samples were ultimately included in the longitudinal ctDNA analysis in the current study. NGS assays were performed with a custom-designed panel that covered 769 cancer-related genes via the MinerVA platform (Genecast Biotechnology Co., Ltd). Results: Among the cohort, 166 patients harboring targetable mutations in the primary tumor tissue, 15 were preadjuvant ctDNA positive, and among them, only 25% patients treated with adjuvant TKI therapy experienced relapse during follow-up, and patients received ACT or no adjuvant therapy had a relapse rate of 100%. TKI treatment tended to improve RFS compared with no treatment (HR 0.1, P = 0.025), but ACT failed to improve RFS (HR 1.1, P = 0.834). Among all 82 patients receiving adjuvant therapy, the pre- and post-adjuvant therapy postoperative ctDNA status with a negative-negative or positive-negative pattern had similar and favorable RFS ( P = 0.545) but that patients with a positive-positive or negative-positive pattern had significantly inferior RFS than those with a negative-negative pattern (both P < 0.001). For recurrence monitoring, posttreatment ctDNA MRD status had a positive predictive value of 100% and a negative predictive value of 91.8%, and the sensitivity of MRD detection was affected by the organ of relapse. Among positive ctDNA MRD patients, faster increase of ctDNA concentration correlated with more imminent clinical relapse. The median lead time from ctDNA detection to radiological relapse was 273 days. Moreover, ctDNA negativity after relapse was associated with favorable survival. Conclusions: These findings highlight the clinical significance of ctDNA dynamics for the precise whole-course postsurgical management of NSCLC patients.

Utility of circulating tumor DNA (ctDNA) as a predictive biomarker for disease monitoring in patients (pts) with cholangiocarcinoma (CCA) before and during adjuvant chemotherapy (ACT): Sub-analysis of the randomized phase 2 STAMP trial.

4123 Background: Detection of circulating tumor DNA (ctDNA) in blood is a powerful predictive biomarker for identifying molecular residual disease (MRD) post-surgery. Not much is known about the utility of identifying MRD using ctDNA in pts with resected CCA, despite its associated poor prognosis. The STAMP trial (NCT03079427), a randomized phase 2 study comparing adjuvant capecitabine (CAP) to gemcitabine plus cisplatin (GemCis) showed no difference in recurrence-free survival (RFS) and overall survival (OS) between the 2 arms. The goal of this analysis was to evaluate the feasibility of monitoring ctDNA to predict the risk of recurrence in pts with resected CCA. Methods: A total of 254 plasma samples were collected from a cohort of 89 CCA (hilar, N = 43; distal, N = 46) pts post-surgery at 3 separate time points; pre-ACT (baseline, N = 89), on-ACT C5D1 (after 5 cycles of treatment) (N = 88), and on-ACT C8D1 (after 8 cycles of treatment) (N = 77). Longitudinal ctDNA testing was performed using a personalized, tumor-informed ctDNA assay (Signatera, bespoke mPCR-NGS assay). ctDNA results were analyzed and evaluated for its correlation with RFS and OS. Results: At the MRD timepoint (baseline, pre-ACT), 24.7% (22/89) pts were ctDNA-positive, 90.9% (20/22) of whom recurred demonstrating a trend for shorter RFS (HR = 1.7, 95%Cl 0.98-2.8, p = 0.069) compared to ctDNA-negative pts. All pts who remained ctDNA-positive on-ACT at C5D1 (N = 17) and C8D1 (N = 15), recurred clinically with a significantly shorter RFS (HR = 8.1, 95%Cl 4.3-15, p < 0.0001 and HR = 5.0, 95%Cl 2.7-9.4, p < 0.0001, respectively). The primary cancer site (hilar vs distal) or type of ACT (CAP vs GemCis) had no impact on RFS irrespective of ct-DNA result. Pts who remained ctDNA-negative on ACT (N = 56) showed significantly better RFS compared to pts who remained positive (N = 10, HR = 6.6, 95%Cl 3.1-14, p < 0.001), or who turned positive (N = 11, HR = 5.2, 95%Cl 2.54-10.5, p < 0.001). In multivariate analysis including primary tumor site, sex, pathological grade and ACT regimen, positive ctDNA status at baseline (pre-ACT) remained significant for poor RFS (HR = 1.82, 95%CI 1.04-3.18, p = 0.035). Conclusions: Personalized monitoring of ctDNA both pre and on-ACT is feasible and predictive for recurrence in pts with resected CCA. To our best knowledge, this is one of the first reports presenting the clinical implication of ctDNA monitoring on ACT, in pts with CCA. Our findings suggest ctDNA monitoring may help optimize clinical decision-making in pts with resected CCA. Larger prospective studies would be needed to validate the findings of this study. Clinical trial information: NCT03079427 .

Enhanced detection of molecular residual disease (MRD) by tumor-informed and tumor-agnostic variants in circulating tumor DNA (ctDNA).

e15586 Background: Liquid biopsies have been mainly used for cancer detection, non-invasive tumor genotyping and disease surveillance. Highly sensitive liquid biopsy assays have been developed that can now be applied to detect and characterize MRD. Several techniques have been developed for the detection of MRD, mainly by tracking ultra-low-frequency somatic tumor mutations in cell-free DNA. Methods: NGS using a 1123-gene-panel were performed on the tumor tissue and white blood cells which was designed for pan-cancer covering 1.8Mbp human genome. Moreover, we used a self-designed pan-cancer panel covering 208 kb and increasing 20 or less mutations individually known to be present in the surgical tissue based on the fixed panel for postoperative plasma or follow-up plasma. In Our study, plasma were collected from 137 healthy donors and UMI sequencing with 208 kb panel for built an database of cfDNA background noise. Furthermore, we constructed two different databases of CH(clonal hematopoietic) variants and negative-background noise with 2300+ cancer patients and 1000+ tumor-plasma paired data respectively. In this way, this approach not only could track selected tumor-specific mutations, but also provided with new variants using tumor-agnostic. MRD-positive status was defined as at least one variants in the plasma. Results: To test sensitivity and specificity of the minimal residual disease detection (MRD) method, we used 208kb panel data for plasma samples from 137 healthy samples and 150 samples with CRC untreatment. The result showed an area under the curve (AUC) of 0.95, with sensitivity of 92% and specificity of 98%. To test the limit of detection of 208 kb panel, we sequenced six different dilution levels (5%, 1%, 0.5%, 0.1%, 0.05% and 0.01%) with a positive control sample containing 98 known mutations. The result showed the detection limitation of 208 kb panel achieved 0.02%. We retrospectively analyzed 3000+ patients with colorectal cancer, 35 of whom met the eligibility criteria were enrolled in this study: 1) With stage II-III CRC undergoing radical surgery; 2)Blood specimens were collected on postoperative 7 days to 3 months; 3)Follow-up duration was 2 years or relapsed; 4)Enough samples for targeting sequencing. Twelve (34%) patients with MRD+,which all patients recurred while 23(66%) patients were MRD-, which 22/23 patient did not experience local recurrence or metastasis. The Kaplan-Meier estimates showed that the positivity of ctDNA was of high predictive value for relapse (HR 21, 95%CI 18.3-28.9;P < 0.001). Conclusions: In this study, combination of tumor-informed and tumor-agnostic strategies can effectively distinguish healthy and cancer patients. Meanwhile,our study highlights that postoperative ctDNA status can effectively detect MRD and identify patients with a high risk of relapse.

CIRCULATING TUMOR DNA (CTDNA) STATUS AND CLINICAL OUTCOMES IN PATIENTS (PTS) WITH PREVIOUSLY UNTREATED DIFFUSE LARGE B‐CELL LYMPHOMA (DLBCL) IN THE POLARIX STUDY

CIRCULATING TUMOR DNA (CTDNA) STATUS AND CLINICAL OUTCOMES IN PATIENTS (PTS) WITH PREVIOUSLY UNTREATED DIFFUSE LARGE B‐CELL LYMPHOMA (DLBCL) IN THE POLARIX STUDY

HPV integration as a prognostic biomarker for metastatic anal cancer: A next-generation sequencing ctDNA-based approach.

3525 Background: Anal cancer is an HPV-associated malignancy with increasing prevalence in the United States. Immune checkpoint blockade demonstrates limited efficacy in patients with metastatic squamous cell cancer of the anal canal (mSCCA), and no prognostic or predictive biomarkers have demonstrated clinical utility in this population. Available circulating tumor DNA (ctDNA) assays have utilized ddPCR methodologies to report only the most common HPV types (HPV-16 and HPV-18) and HPV copy numbers. To extend the utility of an HPV ctDNA assay, we created a novel next-generation sequencing HPV ctDNA assay to analyze HPV integration as a prognostic biomarker for patients with mSCCA. Methods: Using an IRB-approved protocol, ctDNA isolated from the plasma of patients with mSCCA was sequenced using a 1.4Mb hybrid-capture target-enrichment panel covering the whole genome sequences of all 193 HPV types. HPV type, HPV copy number, and HPV integration status/locus were determined using a bioinformatic pipeline developed in-house. A Fisher's exact test was used to compare radiographic and ctDNA changes in response to systemic treatment. Unpaired t-tests were used to compare demographics between patients with HPV integration (I) or no integration (NI) status in the ctDNA. Median survival was estimated (Kaplan Meier) and compared according to integration status using a log-rank test. Results: 68 plasma samples from 27 patients with mSCCA were analyzed. While HPV-16 was detected in ctDNA of all 27 patients, five patients had additional oncogenic HPV types [HPV-18 (2), HPV-45 (2), HPV-73 (1)]. Radiographic changes in metastatic disease volume were concordant with HPV copy number changes in the ctDNA (odds ratio 12.2, 95% confidence interval (CI) 2.0-75; p = .007). HPV integration events were detected in the ctDNA for 16 (59%) patients (median 2, range 1-7 unique events per patient). For the 23 patients with mSCCA who received anti-PD1 immunotherapy (15 I, 8 NI), there were no differences in age (60 vs 59 years, p = .88) or prior lines of treatment (3.0 vs 3.5, p = .72). There was a trend towards improved median progression-free survival for NI ctDNA status (5.8 vs 3.2 months, hazard ratio (HR) 1.8, 95% CI .71-4.5). Relative to NI status, I status was associated with worse overall survival rates (OS) (22.5 vs 44.9 months, HR 5.0, 95% CI 1.5-17, p = .005). Conclusions: Detection of HPV integration status for all HPV types is feasible using a ctDNA-based approach for patients with SCCA. HPV integration was prognostic for worse OS in patients with mSCCA. Identification of HPV integration as a novel prognostic biomarker for survival outcomes in mSCCA warrants further evaluation in larger clinical trials.

Predicting role of circulating tumor DNA and blood-based tumor mutational burden in esophageal squamous cell carcinoma receiving chemoradiotherapy combined with toripalimab: Exploratory analyses from a phase II trial (EC-CRT-001).

4056 Background: EC-CRT-001 is a phase II trial investigating the efficacy of toripalimab (an anti-PD1 antibody) combined with definitive chemoradiotherapy (CRT) in locally advanced esophageal squamous cell carcinoma (ESCC) and the results demonstrated encouraging antitumor activity and acceptable toxicity. This exploratory analysis was performed to evaluate the role of circulating tumor DNA (ctDNA) and blood-based tumor mutational burden (bTMB) for efficacy prediction. Methods: Forty-two patients from the phase II trial (NCT04005170) who received toripalimab (240 mg every 3 weeks for up to 1 year, or disease progression, or unacceptable toxicity) combined with definitive CRT (irradiation with 50.4 Gy in 28 fractions concurrent with 5 cycles of weekly paclitaxel in 50 mg/m2 and cisplatin in 25 mg/m2) were enrolled. Plasma samples were collected before, during, and after CRT. Targeted next-generation sequencing was performed on a total of 118 plasma samples and 35 matched tumor samples using a panel covering 474 cancer-associated genes. Results: Twenty-six (62%) of 42 patients achieved clinical complete response (cCR) at three months after CRT. ctDNA was detected in 29 (72.5%) of 40 patients at baseline. The positive rate of ctDNA decreased to 43.9% (20/41) during CRT and continued to descend to 27.0% (10/37) at the completion of CRT. A higher cCR rate was observed in patients with negative during-CRT ctDNA compared to those with detectable ctDNA (82.6% vs. 38.9%, P = 0.008). Patients with post-CRT detectable ctDNA also had poorer cCR rate (30.0% vs. 77.8%, P = 0.017). Within a median follow-up of 24.0 months (IQR 13.7-27.9), the median progression-free survival (PFS) was 12.2 months (95%CI: 8.4-16.0) and median overall survival (OS) was not reached for the whole cohort. Patients with during-CRT detectable ctDNA had shorter PFS compared to ctDNA-negative patients (HR = 2.57, 95%CI: 1.18-5.60, P = 0.014). Similarly, patients with post-CRT detectable ctDNA also had a significantly increased risk of disease progression (HR = 2.88, 95%CI: 1.21-6.83, P = 0.012) and death (HR = 3.67, 95%CI: 1.41-9.55, P = 0.004). Moreover, patients with higher bTMB ( > 1) detected during CRT were associated with a favorable OS (HR = 0.33, 95%CI: 0.13-0.88, P = 0.027). Improved PFS was also observed in those patients with higher post-CRT bTMB ( > 3) (HR = 0.28, 95%CI: 0.08-0.96, P = 0.042). Conclusions: Negative ctDNA status and higher bTMB during or after CRT were associated with better tumor response and favorable survival in ESCC patients who underwent definitive CRT combined with toripalimab. Dynamic ctDNA has great potential in predicting efficacy and risk of disease progression in ESCC. Clinical trial information: NCT04005170 .

Use of circulating tumor DNA analysis to predict recurrence and need for adjuvant chemotherapy in stage I-IV colorectal cancer.

e15537 Background: Circulating tumor DNA (ctDNA) has emerged as a biomarker that can define the risk of recurrence after curative-intent surgery for patients with colorectal cancer (CRC). However, beyond the predictive power of postoperative ctDNA detection, the efficacy and potential limitations of ctDNA detection urgently need to be fully elucidated in a large cohort of CRC. Methods: We enrolled 309 patients with stages Ⅰ to Ⅳ CRC who underwent definitive surgery. Tumor tissues from those patients were sequenced by a custom-designed next-generation sequencing (NGS) panel to identify somatic mutations, and plasma was analyzed using ctDNA-based molecular residual disease (MRD) assay which integrated tumor genotype–informed and tumor genotype–naïve ctDNA analysis. Results: ctDNA was detected after surgery in 5.4%, 13.8%, 15% and 30% of patients with stage Ⅰ, Ⅱ, Ⅲ and Ⅳ disease, respectively, and in 17.5% of all longitudinal samples. Detection of MRD after surgery and in serial plasma samples during follow-up were associated with shorter disease-free survival (DFS) (hazard ratio [HR], 13.17; P < 0.0001 and HR, 14.44; P < 0.0001, respectively) independent of tumor site, stage, and MSI status. Within the population, only 13 patients (6.6%) with longitudinal undetectable MRD recurred, resulting in a negative predictive value of 93.4%. Correspondingly, the positive predictive value of longitudinal detectable MRD was 79.6%, with a median lead time of 10.1 months. Further subgroup analyses revealed that adjuvant therapy did not confer a superior survival for patients with undetectable or detectable MRD. Moreover, lung-only and peritoneal metastases were less detectable by MRD. Conclusions: Postoperative ctDNA status is a strong predictor of recurrence independent of stage and MSI status. Longitudinal undetectable MRD could be used to define the potentially cured population in CRC patients undergoing curative-intent surgery.

Association of tumor-informed minimal residual disease (MRD) with clinical outcomes for muscle invasive bladder cancer (MIBC): A multicenter retrospective real-world analysis.

3050 Background: Muscle invasive bladder cancer is associated with a significant elevated risk of mortality and morbidity due to micro metastatic spread at the time of diagnosis. Accurate biomarkers to guide treatment determination and evaluate response are needed. Previous studies using tumor informed ct-DNA have shown that MRD assessment may be informative in these scenarios. Methods: We conducted a retrospective multicenter analysis of real-world data obtained from commercial testing (Signatera; Natera, Inc.). This cohort of MIBC patients included those who underwent radical cystectomy with a minimum follow up of 90 days. Data were collected on patient demographics, clinical and pathological staging, histologic type, and treatments. Correlation of ct-DNA status with clinical outcomes was analyzed using Cox-regression analysis to evaluate utility of the assay for detecting residual disease post-surgery and radiological recurrence. Results: A total of 109 patients with 545 plasma samples were identified that fit the inclusion criteria. The majority of the patients [79, 72.5%] were male, with a median age of 70 (range: 32-88) years. Most patients had pure urothelial carcinoma [73.4%, 80/109]. Median follow-up was 13.63 (range: 3-89.04) months. On analyzing the first time point post-surgery (median 54 days), 91.55% (65/71) of ct-DNA negative patients at the first post-surgical test remained recurrence free while 65.79% (25/38) of positive patients had recurred at data cut-off. Therefore, ct-DNA positivity at the first post-surgical timepoint was associated with shorter disease-free survival (DFS) (HR=9.9, 95%CI: 4.1-24%, p<0.001). ct-DNA detected imaging recurrence with a median lead time of 61 days (range: 12-381). Similarly, on analyzing ct-DNA positivity at any time post-surgery, of those who remained ct-DNA negative (N=63), 96.83% (61/63) showed no evidence of recurrent disease. On the other hand, 63.04% (29/46) of pts with ct-DNA positivity at any time after cystectomy recurred and experienced reduced DFS (HR=25, 95%CI: 5.9-103%, p<0.001). The assay’s sensitivity for predicting radiological recurrence at any time post-surgery was 93.55%. Notably, in the multivariate analysis, anytime ct-DNA positivity after cystectomy was found to be the only significant risk factor associated with shorter DFS (HR=23.07, 95%CI: 5.1-104.4, p<0.001). Conclusions: In this initial analysis of real-world data, MRD detection via tumor-informed ct-DNA testing was highly prognostic of patient outcomes post-surgery and may guide adjuvant treatment decisions. Future studies in this setting will explore the utility of ct-DNA testing as a predictive biomarker in response to systemic therapy.

Personalized circulating tumor DNA (ctDNA) analysis in predicting residual disease after neoadjuvant chemoradiotherapy (nCRT) for esophageal squamous cell carcinoma (ESCC).

e16062 Background: For ESCC patients who receive nCRT, high pathological complete response (pCR) rates could be achieved. A “Watch and Wait” strategy has been proposed for patients with clinical complete response (cCR) after nCRT. However, clinical assessments would inevitably misclassify non-pCR as cCR. We previously showed that ctDNA analysis with tumor-naïve fixed panel could improve the prediction of cCR. This study further investigates the value of personalized ctDNA analysis in predicting tumor response and residual disease after nCRT in ESCC. Methods: This is a side study of the prospective, diagnostic preSINO trial (NCT03937362). After completion of nCRT, patients underwent one or two clinical response evaluations (CREs) before planned surgery. CRE‐1 was 4–6 weeks after nCRT. CRE‐2 was 10–12 weeks after nCRT for patients without histological evidence of residual tumor at CRE-1. Individual tumors and matched white blood cells at baseline (B0) were whole-exome sequenced and somatic mutations were identified. Serial plasma samples were analyzed by brPROPHET tracking up to 50 somatic mutations at three timepoints: B0, CRE-1, CRE-2. This assay was developed to detect residual disease with a limit of detection of 0.004% and sample-level specificity of greater than 99% in the analytical validation. Residual disease was also detected and quantified through TNFP in the same samples for a head-to-head comparison. We correlated post-nCRT ctDNA status with pCR and major locoregional residual disease (MLRD) (>10% residual cancer or any residual nodal disease). Results: At this interim analysis, 55 ESCC patients (stage III, 87%) who received per-protocol treatments had sufficient sample to test ctDNA. The pCR rate was 27 % (15/55). 98% (2918/2966) of variants selected for patient-specific panel design were private to a specific patient, only 3% (92/2966) of variants were included in the fixed panel. After nCRT, ctDNA was detected by brPROPHET prior to nCRT in 100% of samples (n=55), significantly higher than that by TNFP assay (73%, 40/55). ctDNA was detected in 55% (30/55) of patients at CRE-1, 43% (10/23) at CRE-2. Importantly, the brPROPHET achieved higher sensitivity than TNFP in predicting non-pCR and MLRD owing to its ability to detect extremely low ctDNA fractions (to 1.14 x 10-5). Moreover, combining brPROPHET with CREs could further improve the sensitivity. Conclusions: Personalized ctDNA analysis can supplement the clinical assessment to decrease the false negative rate in identifying patients with residual disease after nCRT, and might become useful in the “Watch and Wait” strategy for the management of ESCC. Clinical trial information: NCT03937362 . [Table: see text]

Early serial circulating tumor DNA sequencing predicts the efficacy of chemohormonal therapy in patients with metastatic hormone-sensitive prostate cancer

Chemohormonal therapy is a standard treatment for metastatic hormone-sensitive prostate cancer (mHSPC); however, there are no biomarkers to guide clinical decisions regarding therapeutic options. We aimed to evaluate the clinical utility of serial circulating tumor DNA (ctDNA) sequencing in early prediction of the efficacy of chemohormonal therapy in patients with mHSPC. We conducted a retrospective observational study of 66 patients with mHSPC receiving chemohormonal therapy who underwent serial targeted gene-panel ctDNA sequencing. Peripheral blood samples were collected before treatment and after one cycle of chemotherapy. Kaplan–Meier and log-rank analyses were used to analyze the association between ctDNA status and disease progression-free survival. Serial changes in the ctDNA fraction and genetic alterations were also observed. After one cycle of chemotherapy, 23 (34.8%) patients displayed elevated ctDNA levels, whereas the other patients (65.2%, n = 43) did not. The median time to castration resistance in the group with reduced ctDNA levels was significantly longer than that in the group with increased ctDNA levels (17.70 vs. 8.43 months [mo], p < 0.001). Interestingly, patients with de novo alterations in homologous recombination pathway genes after treatment experienced a shorter time to castration resistance than that experienced by the remaining patients (8.02 vs. 13.20 mo, p = 0.011). The increased ctDNA levels or de novo alterations detected in homologous recombination pathway genes are a harbinger of disease progression. Early serial ctDNA sequencing could aid clinicians in making accurate treatment decisions.

Phase II trial of delta-tocotrienol in neoadjuvant breast cancer with evaluation of treatment response using ctDNA

Neoadjuvant treatment of breast cancer is applied to an increasing extent, but treatment response varies and side effects pose a challenge. The vitamin E isoform delta-tocotrienol might enhance the efficacy of chemotherapy and reduce the risk of side effects. The aim of this study was to investigate the clinical effect of delta-tocotrienol combined with standard neoadjuvant treatment and the possible association between detectable circulating tumor DNA (ctDNA) during and after neoadjuvant treatment with pathological treatment response. This open-label, randomized phase II trial included 80 women with newly diagnosed, histologically verified breast cancer randomized to standard neoadjuvant treatment alone or in combination with delta-tocotrienol. There was no difference in the response rate or frequency of serious adverse events between the two arms. We developed a multiplex digital droplet polymerase chain reaction (ddPCR) assay for the detection of ctDNA in breast cancer patients that targets a combination of two methylations specific for breast tissue (LMX1B and ZNF296) and one cancer specific methylation (HOXA9). The sensitivity of the assay increased when the cancer specific marker was combined with the ones specific to breast tissue (p < 0.001). The results did not show any association between ctDNA status and pathological treatment response, neither at midterm nor before surgery.

Recurrence Prediction by Circulating Tumor DNA in the Patient with Colorectal Liver Metastases After Hepatectomy: A Prospective Biomarker Study.

The recurrence rate after hepatic resection of colorectal liver metastases (CRLM) remains high. This study aimed to investigate postoperative circulating tumor DNA (ctDNA) based on ultra-deep next-generation sequencing (NGS) to predict patient recurrence and survival. Using the high-throughput NGS method tagged with a dual-indexed unique molecular identifier, named the CRLM-specific 25-gene panel (J25), this study sequenced ctDNA in peripheral blood samples collected from 134 CRLM patients who underwent hepatectomy after postoperative day 6. Of 134 samples, 42 (31.3%) were shown to be ctDNA-positive, and 37 resulted in recurrence. Kaplan-Meier survival analysis showed that disease-free survival (DFS) in the ctDNA-positive subgroup was significantly shorter than in the ctDNA-negative subgroup (hazard ratio [HR], 2.96; 95% confidence interval [CI], 1.91-4.6; p < 0.05). When the 42 ctDNA-positive samples were further divided by the median of the mean allele frequency (AF, 0.1034%), the subgroup with higher AFs showed a significantly shorter DFS than the subgroup with lower AFs (HR, 1.98; 95% CI, 1.02-3.85; p < 0.05). The ctDNA-positivepatients who received adjuvant chemotherapy longer than 2 months showed a significantly longer DFS than those who received treatment for 2 months or less (HR, 0.377; 95% CI, 0.189-0.751; p < 0.05). Uni- and multivariable Cox regression indicated two factors independently correlated with prognosis: ctDNA positivity and no preoperative chemotherapy. The study demonstrated that ctDNA status 6 days postoperatively could sensitively and accurately predict recurrence for patients with CRLM using the J25 panel.

NK cell activity and methylated HOXA9 ctDNA as prognostic biomarkers in patients with non-small cell lung cancer treated with PD-1/PD-L1 inhibitors

BackgroundPD-1/PD-L1 inhibitors have improved survival for patients with non-small cell lung cancer (NSCLC). We evaluated natural killer cell activity (NKA) and methylated HOXA9 circulating tumor DNA (ctDNA) as prognostic biomarkers in NSCLC patients treated with PD-1/PD-L1 inhibitors.MethodsPlasma was prospectively collected from 71 NSCLC patients before treatment with PD-1/PD-L1 inhibitors and before cycles 2–4. We used the NK Vue® assay to measure the level of interferon gamma (IFNγ) as a surrogate for NKA. Methylated HOXA9 was measured by droplet digital PCR.ResultsA score combining NKA and ctDNA status measured after one treatment cycle had a strong prognostic impact. Group 1 had IFNγ < 250 pg/ml and detectable ctDNA (n = 27), group 2 consisted of patients with either low levels of IFNγ and undetectable ctDNA or high levels of IFNγ and detectable ctDNA (n = 29), group 3 had IFNγ ≥250 pg/ml and undetectable ctDNA (n = 15). Median OS was 221 days (95% CI 121–539 days), 419 days (95% CI 235–650 days), and 1158 days (95% CI 250 days—not reached), respectively (P = 0.002). Group 1 had a poor prognosis with a hazard ratio of 5.560 (95% CI 2.359–13.101, n = 71, P < 0.001) adjusting for PD-L1 status, histology, and performance status.ConclusionsCombining NKA and ctDNA status after one cycle of treatment was prognostic in patients with NSCLC treated with PD-1/PD-L1 inhibitors.

Early Detection of Molecular Residual Disease and Risk Stratification for Stage I to III Colorectal Cancer via Circulating Tumor DNA Methylation

Detection of molecular residual disease and risk stratification as early as possible may improve the treatment of patients with cancer. Efficient pragmatic tests are therefore required. To measure circulating tumor DNA (ctDNA) with 6 DNA methylation markers in blood samples and to evaluate the association of the presence of ctDNA with colorectal cancer (CRC) recurrence throughout the disease course. In this multicenter prospective longitudinal cohort study performed from December 12, 2019, to February 28, 2022, 350 patients with stage I to III CRC were recruited from 2 hospitals for collection of blood samples before and after surgery, during and after adjuvant chemotherapy, and every 3 months for up to 2 years. A multiplex, ctDNA methylation, quantitative polymerase chain reaction assay was used to detect ctDNA in plasma samples. A total of 299 patients with stage I to III CRC were evaluated. Of 296 patients with preoperative samples, 232 (78.4%) tested positive for any of the 6 ctDNA methylation markers. A total of 186 patients (62.2%) were male, and the mean (SD) age was 60.1 (10.3) years. At postoperative month 1, ctDNA-positive patients were 17.5 times more likely to relapse than were ctDNA-negative patients (hazard ratio [HR], 17.5; 95% CI, 8.9-34.4; P < .001). The integration of ctDNA and carcinoembryonic antigen tests showed risk stratification for recurrence with an HR of 19.0 (95% CI, 8.9-40.7; P < .001). Furthermore, ctDNA status at postoperative month 1 was strongly associated with prognosis in patients treated with adjuvant chemotherapy of different durations and intensities. After adjuvant chemotherapy, ctDNA-positive patients had a significantly shorter recurrence-free survival than did the ctDNA-negative patients (HR, 13.8; 95% CI, 5.9-32.1; P < .001). Longitudinal ctDNA analysis after the postdefinitive treatment showed a discriminating effect in that ctDNA-positive patients had poorer recurrence-free survival than did the ctDNA-negative patients (HR, 20.6; 95% CI, 9.5-44.9; P < .001). The discriminating effect was enhanced (HR, 68.8; 95% CI, 18.4-257.7; P < .001) when ctDNA status was maintained longitudinally. Postdefinitive treatment analysis detected CRC recurrence earlier than radiologically confirmed recurrence, with a median lead time of 3.3 months (IQR, 0.5-6.5 months). The findings of this cohort study suggest that longitudinal assessment of ctDNA methylation may enable the early detection of recurrence, potentially optimizing risk stratification and postoperative treatment of patients with CRC.